This proposed collaborative project will investigate fundamental processes driving chordate embryogenesis. The project will combine the skills and expertise of two research groups: one that works in the area of developmental biology, and the other in the area of image analysis and computer vision. The goal of the project is take a whole-embryo embryo approach to investigating morphogenesis in live embryos in all 4 dimensions (x,y, z and t). Specifically, we will collect and analyze confocal microscopy images to derive quantitative data on the division, shape, volume and movements of all cells in both selected developing organs and in whole embryos. This project presents many challenges in sample selection and labeling, image capture, and image analysis and visualization. Most model organisms are fundamentally unsuited to the challenge of capturing the shape and movement of every cell in an entire tissue. Those that are suitable typically are distantly related to the chordates, and thus lack organs and tissues relevant to human health and disease. The ascidians (sea squirts), however, are close relatives of the vertebrates that combine a chordate body plan with a small and simple embryonic architecture suitable for imaging in toto. The data to be collected in such observations, particularly at the high resolution needed to observe cellular behavior, will be enormous. The extraction of large-scale quantitative information from timelapse image sets poses substantial challenges in terms of image segmentation and analysis. We have already made significant progress in segmenting 3D ascidian images and further refinements of these approaches are proposed here. The segmented data will allow the quantitative analysis of many complex cell behaviors, including the fundamental changes in cell shape and motility driving gastrulation, neurulation and convergent extension. These analyses will lead to specific hypotheses regarding the cellular and molecular machinery driving morphogenesis. To further investigate the molecular basis for these cellular phenomena, we will also extend our imaging and analysis efforts to investigate the dynamic patterns of subcellular localization of several proteins with key roles in morphogenesis.